Pathological features and molecular signatures of early olfactory dysfunction in 3xTg‐AD model mice

Abstract Background Olfactory dysfunction is known to be an early manifestation of Alzheimer's disease (AD). However, the underlying mechanism, particularly the specific molecular events that occur during the early stages of olfactory disorders, remains unclear. Methods In this study, we utilized transcriptomic sequencing, bioinformatics analysis, and biochemical detection to investigate the specific pathological and molecular characteristics of the olfactory bulb (OB) in 4‐month‐old male triple transgenic 3xTg‐AD mice (PS1M146V/APPSwe/TauP301L). Results Initially, during the early stages of olfactory impairment, no significant learning and memory deficits were observed. Correspondingly, we observed significant accumulation of amyloid‐beta (Aβ) and Tau pathology specifically in the OB, but not in the hippocampus. In addition, significant axonal morphological defects were detected in the olfactory bulb, cortex, and hippocampal brain regions of 3xTg‐AD mice. Transcriptomic analysis revealed a significant increase in the expression of neuroinflammation‐related genes, accompanied by a significant decrease in neuronal activity‐related genes in the OB. Moreover, immunofluorescence and immunoblotting demonstrated an activation of glial cell biomarkers Iba1 and GFAP, along with a reduction in the expression levels of neuronal activity‐related molecules Nr4a2 and FosB, as well as olfaction‐related marker OMP. Conclusion In sum, the early accumulation of Aβ and Tau pathology induces neuroinflammation, which subsequently leads to a decrease in neuronal activity within the OB, causing axonal transport deficits that contribute to olfactory disorders. Nr4a2 and FosB appear to be promising targets for intervention aimed at improving early olfactory impairment in AD.


| INTRODUC TI ON
Alzheimer's disease (AD) is the most prevalent neurodegenerative disease characterized by the accumulation of amyloid-beta (Aβ)   and Tau proteins, leading to cognitive decline and memory loss. 1 However, even before the onset of cognitive impairment, individuals with AD commonly experience significant olfactory impairment, indicating that olfactory dysfunction is an early feature of the disease. 2 Unfortunately, the challenge of obtaining brain tissue samples from individuals with early-stage anosmia has hindered the identification of biomarkers associated with early olfactory impairment in AD.4][5] This approach provides valuable insights into the relationship between olfactory dysfunction and the early diagnosis and treatment of AD.
Our previous studies have shown that olfactory dysfunction can be used as an early screening tool, 6 but the specific mechanism remains unclear.It has been reported that neuropathological markers of AD, such as Aβ and Tau pathology, are present in areas associated with olfactory function, particularly the olfactory bulb (OB), in aging and at autopsy of different neurodegenerative diseases. 7OB is also believed to be an entry point for potential pathogens to enter and spread throughout the brain. 8,9The presence of Tau and α-synuclein (αSyn) histopathology in the OB has been reported in the early stages of AD, Lewy body disease (LBD), Parkinson's disease (PD), and multiple system atrophy (MSA). 10,113][14] These findings provide a potential model for understanding the pathogenesis of olfactory-mediated neurodegenerative diseases, including AD and LBD.Furthermore, the presence of Aβ pathology in the OB may serve as an indicator of AD diagnosis.Studies have shown a correlation between Aβ in the OB and the overall amyloid phase and Braak neurofibrillary tangle phase in the brain. 7,11Overall, these findings contribute to our understanding of the role of olfactory dysfunction in neurodegenerative diseases and highlight the potential for using olfactory markers in diagnostics.
Triple transgenic AD mice (3xTg-AD) (Stock No: 34830, 129S4. CgTg [APPSwe, tauP301L] 1LfaPsen1tm1Mpm/Mmjax) began to exhibit learning and memory deficits at 6.5 months of age 15 and olfactory dysfunction at 3-5 months of age. 3 Here, we identified 4-month-old 3xTg-AD mice with pronounced olfactory dysfunction, but without significant impairment in learning and memory, as a suitable model for investigating biomarkers of early olfactory dysfunction.Correspondingly, significant Aβ and Tau pathology was observed in the olfactory bulb (OB) but not in the hippocampus of 4-month-old 3xTg-AD mice.However, significant axonal morphological defects were observed from olfactory bulb to hippocampus in 3xTg-AD mice.Transcriptomic analysis revealed genes related to neuroinflammation were significantly activated, while genes related to neuronal activity were significantly decreased.Validation experiments confirmed the glial cell markers Iba1 and GFAP were significantly activated, while neuronal activity-related molecules Nr4a2 and FosB were significantly decreased within the OB.Nr4a2 and FosB appear to be promising biomarkers and potential targets for intervention in cases of early olfactory dysfunction.

| Olfactory impairment predates cognitive memory impairment in 3xTg-AD mice
In order to detect olfactory and learning and memory deficits in 3xTg-AD mice at the age of 3-4 months, several behavioral tests were performed, such as buried food test, cookie-finding test, and Morris water maze test (Figure 1).The buried food test showed that 3xTg-AD mice had a longer latency to find the buried food than WT mice (Figure 1B,C).The cookie-finding test showed that 3xTg-AD mice entered the wrong arms more often than wildtype mice and had a longer latency ratio to first enter the correct arm than wild-type mice (Figure 1D-F).However, in the water maze test, it was observed that 4-month-old 3xTg-AD mice did not display any learning impairment in comparison to wild-type mice during the training phase (Figure 1G).Additionally, during the memory retrieval stage, there were no significant differences observed in terms of the latency to find the platform, the number of crossings over the platform, or the distance traveled These findings indicate that 3xTg-AD mice exhibit olfactory dysfunction while showing no significant impairments in learning and memory, which is in line with the development of behavioral disorders observed in patients, olfactory dysfunction occurs in the early stages of Alzheimer's disease (AD). 16

| Pathological changes of Tau and Aβ were observed in the olfactory bulb of 4-month-old 3xTg-AD mice
In order to study the pathological changes of Tau and Aβ in the olfactory bulb (OB) of 3xTg-AD mice, we conducted an examination of Tau protein levels and its phosphorylation sites, P-APP.
We discovered that Tau5, Ser262, Thr231, and Ser396 were significantly elevated in the OB (Figure 2).Additionally, we observed increased levels of P-APP and 4G8 in the OB, but not in the hippocampus (Figure 3).These findings align with our behavioral measurements and provide evidence that increased Tau and Aβ pathology may contribute to olfactory dysfunction in 3xTg-AD mice.

| The significant axonal morphological defects were detected in the olfactory bulb, cortex, and hippocampal brain regions of 4-month-old 3xTg-AD mice
Since axons and synapses are crucial for neural electrical signal transduction, we first examined microtubule-associated protein 2 (MAP2) and found that MAP2 was significantly reduced in the olfactory bulb, cortex, and hippocampus of 3xTg-AD mice, along with disrupted axonal morphology (Figure 4A,D).To evaluate synaptic function in mice, we examined synapse-associated proteins (Glun1, Glun2B, and SYN1).Western blot showed that synapse-associated proteins SYN1 showed a slight decrease in hippocampus, and other synapse-associated proteins did not change significantly in OB and hippocampus (Figure 4B,C,E,F).These findings suggest that synapseassociated protein expression remained relatively stable in the OB and hippocampus of the 4-month-old 3xTg-AD mice.These results suggest that the destruction of axons may affect the transmission of olfactory signals through the OB to the hippocampus.

| Transcriptome analysis revealed significant dysregulation of neuronal activity and immune inflammation-related processes in the olfactory bulb of 3xTg-AD mice
Transcriptomics found 540 genes that were differentially expressed (DEGs) in the OB of the 3xTg-AD mice, with 220 upregulated and 200 down-regulated (Figure 5A,B).Gene ontology (GO) analysis showed that DEGs were mainly involved in immune system processes, transcription regulation, and developmental process (Figure 5C).Reactome annotations analysis revealed that DEGs were mainly enrichment in immune system, signal transduction, nervous system, developmental process, cell cycle, and DNA replication (Figure 5D).KEGG enrichment analysis showed that DEGs were mainly involved in signal transduction, nervous system, immune system, sensory system, and immune disease (Figure 5E).
Overall, the analysis revealed changes in neuronal transcriptional activity regulation and immune inflammatory systems in the OB of the 3xTg-AD mice.
Specifically, the DEGs involved in neuronal transcriptional activity mainly include Mnd1, Nr1i3, Macc1, Npas4, Atf3, Nr4a2, Junb, FosB, Egr3, Nr4a3, Egr4 and were all down-regulated in the OB of 3xTg-AD mice (Figure 5B).The DEGs related to immune inflammation  5F).Additionally, we found that the transcription levels of neuroinflammation-related genes IL-6, TNFα, Iba1, and GFAP were significantly elevated in the OB of the 3xTg-AD mice (Figure 5G).This provides further evidence of altered neuronal activity regulation and increased inflammatory response in the OB of these mice.

| The expression of glial cell markers Iba1 and GFAP in brain tissue of 4-month-old 3xTg-AD mice
The activation of glial cells, specifically astroglia and microglia, is closely associated with neuroinflammation.To assess this activation, we measured the levels of the astrocyte marker GFAP and the microglia marker Iba1 using Western blotting and immunofluorescence techniques.The results demonstrated significant activation of both GFAP and Iba1 in the olfactory bulb of 4-month-old 3xTg-AD mice (Figure 6A-F).Interestingly, in the 3xTg-AD hippocampus, there was a significant increase in Iba1 expression, while GFAP expression was no significantly changed, and significant activation was observed only in the CA3 region (Figure 6G-L).These findings indicate glial cells activation in both the olfactory bulb and hippocampus of 4-month-old 3xTg-AD mice.

| The expression of neuronal activity-related molecules Nr4a2 and FosB in the OB of 4-month-old 3xTg-AD mice
Nr4a2 and FosB are transcription factors known to have essential roles in the regulation of neuronal activity. 17Specifically, chronic inflammation, induced by lipopolysaccharide (LPS) stimulation, has been associated with damage to neuronal and synaptic functions.
Activation of Nr4a2 has demonstrated protective effects against the neurotoxic effects caused by chronic inflammation, potentially due to its ability to mitigate damage to neuronal and synaptic functions.Using immunoblotting and immunofluorescence experiments, we observed a significant reduction in the expression of Nr4a2 and FosB, as well as a decrease in colocalization with the olfactory biomarker OMP (Figure 7A-F).Overall, our findings suggest that the dysregulation of Nr4a2 and FosB may lead to impaired neuronal activity in the olfactory system, potentially resulting in olfactory dysfunction.
Additionally, odor receptors represent the largest subfamily of G protein-coupled receptors and play a vital role in olfactory signal transduction.β-arrestin1 (Arrb1) and β-arrestin2 (Arrb2) not only regulate G protein-coupled receptors (GPCRs) endocytosis and desensitization but also contribute to the initiation of alternative signaling pathways, thereby participating in the regulation of olfactory function. 18,19Our data showed a significant decrease in the mRNA levels of Arrb1 and Arrb2, which could potentially impact olfactory signaling by regulating GPCRs (Figure 7G).Overall, this study deepens our understanding of the molecular mechanisms underlying olfactory dysfunction and highlights the importance of Nr4a2, FosB, Arrb1, and Arrb2 in maintaining normal neuronal activity and olfactory function (Figure 7H).Further research is needed to elucidate the specific signaling pathways involved and to explore therapeutic strategies targeted at these transcription factors and their associated signaling molecules to potentially restore olfactory function in cases of dysfunction.

| DISCUSS ION
Olfactory dysfunction is an early symptom of Alzheimer's disease (AD) that occurs before cognitive impairment. 20,21However, the current understanding of this dysfunction in AD patients is limited, which hampers the development of biomarkers and targeted drugs for early olfactory dysfunction in AD.This study found that 4-month-old 3xTg-AD mice showed significant olfactory dysfunction despite no impairment in learning and memory function.Additionally, Tau and Aβ pathologies were observed in the 4-month-old 3xTg-AD mice olfactory bulb (OB), although no significant changes were found in the hippocampus.This suggests that 4-month-old 3xTg-AD mice may serve as an ideal animal model for studying the molecular markers of olfactory dysfunction in the early stages of AD.However, significant axonal morphological defects were observed from olfactory bulb to hippocampus in 3xTg-AD mice.Further experiments demonstrated that early Tau and Aβ pathology may trigger inflammation in the brain, leading to reduced expression of Nr4a2 and FosB, resulting in decreased neuronal activity and olfactory disorders (Figure 7H).Therefore, Nr4a2 and FosB may be ultra-early biomarkers and ideal therapeutic targets for olfactory dysfunction.
AD is the most prevalent degenerative disease among the elderly, and its main clinical manifestation is a progressive decline in learning and memory abilities. 1However, one of the earliest signs of the disease is olfactory dysfunction. 20,21The sense of smell, which includes olfactory threshold, odor identification, and odor discrimination, is vital for mammals, and detecting olfactory dysfunction can be used as an early diagnostic tool for AD. 6 The challenge lies in the fact that we can only analyze brain tissue post-mortem, using cognitive scores and pathological grades, to determine the different stages of AD.
Unfortunately, olfactory dysfunction occurs earlier in the disease progression, so we lack corresponding brain tissue samples to understand the underlying molecular events related to this dysfunction.This study suggested that 4-month-old 3xTg-AD mice exhibit impaired sense of smell but no cognitive impairment.Moreover, the characteristic pathological features observed in AD, such as the clustering of Tau and Aβ pathology in the olfactory bulb, were also present in 4-month-old 3xTg-AD mice at this early stage, while the hippocampus did not show obvious symptoms.This finding aligns with the progression of pathology observed in the brains of human AD patients.Therefore, these 4-month-old 3xTg-AD mice may serve as an ideal model for investigating the molecular mechanisms underlying olfactory dysfunction during the early stages of AD.
Transcriptome analysis combined with bioinformatics system revealed the molecular characteristics of olfactory bulb in 4-month-old 3xTg-AD mice and found that DEGs were mainly enriched in immune inflammatory system, neuronal transcriptional activity, and nervous system.AD is a chronic inflammatory disease, and pathways associated with Aβ and Tau production and aggregation as well as inflammation may converge and coordinate the progression of this neurodegenerative disease. 22Here, the production of Aβ and Tau may contribute to chronic inflammatory effects in the olfactory bulb region, which in turn leads to decreased neuronal activity and impaired olfactory signaling.

Olfactory bulb (OB) is an early affected brain region in AD pa-
tients, which has significant inflammatory effects and plays an important role in the onset and progression of AD. 23,24 Iba1 and GFAP are biomarkers of glial cells, and their increased expression indicates the activation of microglia and astrocytes. 25These glial cells are closely related to neuroinflammation, and their levels are significantly increased in multiple brain regions of patients with Alzheimer's disease (AD), including the olfactory bulb. 23In our study, we confirmed that Iba1 expression was increased in the olfactory bulb and hippocampus, GFAP expression was increased in the olfactory bulb of  Nr4a2 is a transcription factor that plays a crucial role in neural development and is expressed in various regions of the central nervous system. 27,28In AD, knocking down Nr4a2 has been found to significantly worsen the pathological symptoms of AD, while activation of Nr4a2 rescued age-related memory decline and reduced neuroinflammation in the brain of mice. 29,30Nr4a2 reduction has also been implicated in depression, where it mediates the decrease in neuronal activity induced by lipopolysaccharide (LPS), leading to the development of depressive symptoms. 17Furthermore, Nr4a2 is associated with age-related macular degeneration, 31 attention deficit hyperactivity disorder, 32 cardiovascular abnormalities, 33 and neuroinflammation in the brains of patients with Parkinson's disease (PD). 34FosB is indeed a member of the Fos family of transcription factors, 35 which is part of the AP-1 transcription factor complex and are involved in regulating gene expression. 36FosB is specifically considered a marker of chronic neuronal activation, 37 while a decrease F I G U R E 5 Transcriptome analysis revealed significant dysregulation of neuronal activity and immune inflammation-related processes in the olfactory bulb of 3xTg-AD mice.(A,B) The heatmap of differentially expressed genes (DEGs; p < 0.05, fold change >1.5) in the OB for 3xTg-AD vs. WT mice.The Z value of gene abundance was plotted in a red-blue color scale, with red and blue indicating increased and decreased protein expression, respectively (A).The red color of the volcano plot represents significantly differentially expressed genes (B).(C-E) Gene ontology (GO; C), reactome annotations (D) and Kyoto Encyclopedia of Genes and Genomes (KEGG; E) enrichment analysis of DEGs were performed.(F,G) Quantitative real-time PCR (qRT-PCR) verification of specific target genes in volcano plot, genes correlated with neuronal activity (F; n = 6 for each group), genes correlated with inflammation (G; n = 6 for each group).Data were shown as mean ± SEM. *p < 0.05, **p < 0.01; ns, not significant.N = 6 for each group.in its expression can indicate low neuronal activity. 17Here, we found that Nr4a2 and FosB are significantly reduced in the olfactory bulb and also in colocalization with OMP, suggesting that Nr4a2 and FosB may contribute to early olfactory dysfunction in AD via affecting neuronal activity.

| CON CLUS IONS
In this study, we found that 4-month-old 3xTg-AD mice can serve as a suitable model to study the molecular signals associated with ultraearly olfactory dysfunction of AD.Significant Aβ and Tau pathology was observed in the olfactory bulb (OB) of 4-month-old 3xTg-AD mice, along with the activation of genes associated with neuroinflammation and the reduction of genes related to neuronal activity, causing axonal transport deficits that contribute to olfactory disorders (Figure 7H).Nr4a2 and FosB are suggested as potential biomarkers for early olfactory dysfunction and may serve as targets for potential interventions.It is important to note that all experiments conducted with these mice adhere to the ethical considerations and animals' well-being. 12

| Cookie-finding test
As in the previous study, 3 mice were given the opportunity to freely explore an eight-arm maze for 5 min/day for a period of five days.After the acclimation period, the mice underwent five days of biscuit seeking trials.These trials were conducted once a day, with the biscuit placed at one end of the arm.The location of the biscuit was changed on a daily basis, ensuring that the mice did not rely on spatial memory to find the biscuit, but rather used their sense of smell.To evaluate the olfactory function of the mice, the number of entries into the wrong arm was recorded daily.

| Buried food test
The food burial experiments conducted in this study followed the same procedures as described in previous studies, 3,38 which involved assessing odor familiarity, food deprivation, and detection on a daily basis.During the experiments, a bedding depth of 3 cm was used, and the food (a 10 mm peanut chocolate square) was buried beneath 1 cm in one of the corners of the cage.The time it took for the mice to find the food, known as the latency, was recorded.If the mice were unable to find the food within 5 min, a latency score of 300 s was recorded, and the test was stopped.Several measures were recorded during this experiment, including the time it took for the mice to first cross the platform, the number of crossings made within 60 s, and the distance and time taken to reach the target quadrant.These measures allowed for an assessment of memory retention and spatial learning abilities in the mice.

| Reverse transcription and real-time quantitative PCR
Total RNA was extracted from mouse brain tissue by TRIzol method and reverse transcribed to produce complementary DNA (Vazyme Biotech Co., Ltd., China).Real-time PCR was performed using 0.8 μL forward and 0.8 μL reverse primers, 1 μL cDNA, 10 μL SYBR Green PCR parent (Yeasen Biotechnology Co., Ltd., China), and 7.4 μL DEPC water.All the PCR primers employed are listed in Table 1.

| Transcriptome and bioinformatics analysis
Transcriptomics comprehensively characterized the olfactory bulb gene expression of 3xTg-AD mice, including RNA purification, reverse transcription, library construction, and sequencing.To gain insights into the biological functions and pathways associated with the differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the WEB-based GEne SeT AnaLysis Toolkit (http:// www.webge stalt.org) were utilized for performing these analyses.

F I G U R E 1
Olfactory impairment predates cognitive memory impairment in 3xTg-AD mice.(A) Behavioral test workflow.(B,C) Buried food test showed that 3xTg-AD mice took longer to find food.(D-F) The cookie-finding test results showed that 3xTg-AD entered the wrong arm more often and took more time to find food.(G) Escape latency to the hidden platform between Days 1 and 6. (H) Swimming pathway traveled to locate the platform on Day 7. (I) The escape latency, (J) number of crossings of the original position of the platform, (K) traveled distance on Day 7. Data were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.N = 16 for each group.mainly included Tnfrsf9 and Il12rb2, which were significantly upregulated in the OB of 3xTg-AD mice (Figure 5B).Consistent with the transcriptional results, we confirmed the down-regulation of key neuronal transcriptional activity factors and up-regulation of inflammation-related factors by qRT-PCR.(Figure

F
I G U R E 2 Pathological changes of Tau were observed in the olfactory bulb of 4-month-old 3xTg-AD mice.(A,B,D,E) Hyperphosphorylated tau (pS262, pT231, pS396, Ps404) and total tau (Tau5) were increased in olfactory bulb (OB; A,B), while no significant changes were observed in the hippocampus (D,E) subset of 4-month-old 3xTg-AD mice compared with age-and sex-matched wild-type mice measured by Western blotting.N = 6 for each group.(C,F) Expression of Tau pathology in the OB (C) and hippocampus (F) of 3xTg-AD mice measured by immunofluorescence staining.

F I G U R E 3
Pathological changes of Aβ-related protein were observed in the olfactory bulb of 4-month-old 3xTg-AD mice.(A,B,D,E) P-APP were increased in olfactory bulb (OB; A,B), while no significant changes were observed in the hippocampus (D,E) subset of 4-month-old 3xTg-AD mice compared with age-and sex-matched wild-type mice measured by Western blotting.N = 6 for each group.(C,F) Expression of Aβ pathology (4G8) in the OB (C) and hippocampus (F) of 3xTg-AD mice measured by immunofluorescence staining.

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month-old 3xTg-AD mice, indicating significant glial activation and neuroinflammation.Arrb1 and Arrb2, members of arrestin/beta-arrestin protein family, are thought to participate in agonist-mediated desensitization of G-protein-coupled receptors and cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals.During the olfactory function, Arrb2 can ameliorate the neuroinflammation, which is considered to be the main factor causing inflammatory response.Previous studies have showed that the expression of Arrb1 and Arrb2 was mutually regulated in mouse models of PD and may have the opposite functions. 26Taken together, Arrb2 plays an important role in neural signal transduction and may affect olfactory function.

F I G U R E 4
The significant axonal morphological defects were detected in the olfactory bulb, cortex, and hippocampal brain regions of 4-month-old 3xTg-AD mice.(A,D) Expression of MAP2 in the OB (A) and cortex, hippocampus (D) of 3xTg-AD mice measured by immunofluorescence staining.(B,C) The protein levels of GluN1, GluN2B, SYN1 in OB were tested by Western blotting (n = 6).(E,F) The protein levels of GluN1, GluN2B, SYN1 in hippocampus were tested by Western blotting (n = 6).Data were shown as mean ± SEM. *p < 0.05; ns, not significant.| 7 of 12 YU et al.
Triple transgenic AD male mice (3xTg-AD) (Stock No: 34830, 129S4.CgTg [APPSwe, tauP301L] 1LfaPsen1tm1Mpm/Mmjax) and wild-type mice were a gift from Prof. Xifei Yang (Shenzhen Center for Disease Control and Prevention).The experimentation was authorized by the Animal Ethics Committee of Jiangnan University (JN.No20230830m0201215[349]).The area in which they were fed maintained a temperature range of 22-26°C, while the relative humidity was kept between 50%-60%, and adequate food and water were ensured, with 12-h light-dark alternating.F I G U R E 6 The expression of glial cell markers Iba1 and GFAP in brain tissue of 4-month-old 3xTg-AD mice.(A-D) Representative immunofluorescence of Iba-1 and GFAP in the OB (n = 5 for each group).(E,F) Expression of Iba1 and GFAP in the OB of 3xTg-AD mice measured by Western blotting (n = 6 for each group).(G-J) Representative immunofluorescence of Iba-1 and GFAP in the hippocampus (n = 5 for each group).(K,L) Expression of Iba1 and GFAP in the hippocampus of 3xTg-AD mice measured by Western blotting (n = 6 for each group).Data were shown as mean ± SEM. *p < 0.05, **p < 0.01; ns, not significant.

First, a series
of learning and training experiments were conducted over six consecutive days.Mice were placed in the water maze from F I G U R E 7 The expression of neuronal activity-related molecules Nr4a2 and FosB in the OB of 4-month-old 3xTg-AD mice.(A,C) Immunofluorescence staining of FosB+ and OMP+ cells in OB.Scale bar, 50 μm (A); statistic results of FosB+ and OMP+ cells numbers in OB (C; n = 5 for each group).(B,D) Immunofluorescence staining of Nr4a2+ and OMP+ cells in OB.Scale bar, 50 μm (B); statistic results of Nr4a2+ and OMP+ cells numbers in OB (D; n = 5 for each group).(E,F) Expression of OMP, FosB, Nr4a2 in the OB of 3xTg-AD mice measured by Western blotting (n = 6 for each group).(G) Quantitative real-time PCR (qRT-PCR) verification of Arrb1, Arrb2 and OMP, genes correlated with neuronal Signaling (G; n = 6 for each group).(H) Diagram of the early accumulation of Aβ and Tau pathology induces neuroinflammation, which subsequently leads to a decrease in neuronal activity within the OB, causing axonal transport deficits that contribute to olfactory disorders.Data were shown as mean ± SEM. *p < 0.05, **p < 0.01.four quadrants and the time it took them to find the underwater platform was recorded.Memory detection experiments were performed on the second day after the learning and training phase.

2
Antibodies used in the Western blotting analysis and their properties.